Thrombomodulin appears to be an endothelial cell specific membrane bound cell surface protein. Recent studies have shown that rabbit endothelium expresses thrombomodulin activity in primary cultures and within the same cell lines even after multiple passages (12). Therefore it appears likely that thrombombomodulin is synthesized by the endothelial cell. Studies will be initiated to demonstrate endothelial cell synthesis of thrombomodulin. Synthesis and expression of thrombomodulin activity will be studied with cells in log phase and following confluence. The expression of thrombomodulin antigen will also be studied in relationship to the cell cycle and in vitro cell aging using flow cytometry procedures. The retention of thrombomodulin activity in continuous culture will be studied in relationship to the loss of other endothelial cell markers such as Factor VIII antigen and angiotensin converting enzyme. The ultrastructural localization of thrombomodulin will be studied by immuno-electron-cytochemistry using ferritin conjugated antithrombomodulin antibodies or by indirect enzyme immunocytochemical techniques. The distribution of thrombomodulin throughout the vasculature will be studied by immunofluorescence and comparisons will be made between large vs. microvessels as well as between microvessels at different tissue sites (i.e., brain vs. lung vs. heart vs. etc.). These studies will provide qualitative information about the organ specificity of thrombomodulin distribution. It is possible that other cell types may have thrombomodulin associated antigen. Of particular interest are the formed elements of the blood and bone marrow which will be investigated by flow cytometry using anti-thrombomodulin antibodies (poly-and monoclonal). These studies will provide the information necessary to initiated studies on the mechanisms controlling thrombomodulin biosynthesis, expression of the antigen and regulation of thrombomodulin's biological activity.